Intercellular and intracellular proximity-based labeling compositions and systems

Abstract

In one aspect, transition metal complexes are described herein having composition and electronic structure for generating reactive labeling intermediates having lifetimes and diffusion radii advantageous for proximity-based labeling of various biomolecular species, including proteins, in intracellular and intercellular environments.

1 . A transition metal complex of Formula I: 
       
         
           
           
               
               
           
         
         wherein M is a transition metal; 
         wherein A, D, E, G, Y and Z are independently selected from C and N; 
         wherein R 3 -R 7  each represent one to four optional ring substituents, each of the one to four optional ring substituents independently selected from the group consisting of alkyl, heteroalkyl, haloalkyl, haloalkenyl, halo, hydroxy, alkoxy, amine, amide, ether, —C(O)O − , —C(O)OR 8 , and —R 9 OH, wherein R 8  is selected from the group consisting of hydrogen and alkyl, and R 9  is alkyl; 
         wherein R 1  is selected from the group consisting of a direct bond, alkylene, alkenylene, cycloaklylene, cycloalkenylene, arylene, heteroalkylene, heteroalkenylene, heterocyclene, and heteroarylene; 
         wherein L is a linking moiety selected from the group consisting of amide, ester, sulfonamide, sulfonate, carbamate, and urea; and 
         R 2  is selected from the group consisting of alkyne, amine, protected amine, azide, hydrazide, aryl, heteroaryl, cycloalkyl, cycloalkenyl, cycloalkylnyl, heterocyclyl, hydroxy, carboxyl, halo, alkoxy, maleimide, —C(O)H, —C(O)OR 8 , —OS(O 2 )R 9 , thiol, biotin, oxyamine, and haloalkyl, wherein R 8  and R 9  are independently selected from the group consisting of alkyl, haloalkyl, aryl, haloaryl, N-succinimidyl, and N-succinimidyl ester; and wherein X −  is a counterion, and n is an integer from 0 to 20. 
       
     
     
         2 . The transition metal complex of  claim 1 , wherein M is a platinum group metal. 
     
     
         3 . The transition metal complex of  claim 2 , wherein M is iridiuim. 
     
     
         4 . The transition metal complex of  claim 1  having an absorption spectrum in the visible region of the electromagnetic spectrum. 
     
     
         5 . The transition metal complex of  claim 1 , wherein R 2  is selected to comprise a moiety for coupling a biomolecule. 
     
     
         6 . The transition metal complex of  claim 5 , wherein the R 2  is a click chemistry moiety. 
     
     
         7 . The transition metal complex of  claim 6 , wherein the click chemistry moiety is selected from the group consisting of BCN, DBCO, TCO, tetrazine, alkyne, and azide. 
     
     
         8 . The transition metal complex of  claim 7 , wherein the biomolecule is an antibody. 
     
     
         9 . The transition metal complex of  claim 1 , wherein the transition metal complex is cell permeable. 
     
     
         10 . The transition metal complex of  claim 1 , having aqueous solubility of 1 μM to 150 μm at 0.2% DMSO in pure water. 
     
     
         11 . A conjugate comprising:
 a transition metal complex coupled to a biomolecular binding agent, wherein prior to coupling to the biomolecular binding agent, the transition metal complex is of Formula I:   
       
         
           
           
               
               
           
         
         wherein M is a transition metal; 
         wherein A, D, E, G, Y and Z are independently selected from C and N; 
         wherein R 3 -R 7  each represent one to four optional ring substituents, each of the one to four optional ring substituents independently selected from the group consisting of alkyl, heteroalkyl, haloalkyl, haloalkenyl, halo, hydroxy, alkoxy, amine, amide, ether, —C(O)O − , —C(O)OR 8 , and —R 9 OH, wherein R 8  is selected from the group consisting of hydrogen and alkyl, and R 9  is alkyl; 
         wherein R 1  is selected from the group consisting of a direct bond, alkylene, alkenylene, cycloaklylene, cycloalkenylene, arylene, heteroalkylene, heteroalkenylene, heterocyclene, and heteroarylene; 
         wherein L is a linking moiety selected from the group consisting of amide, ester, sulfonamide, sulfonate, carbamate, and urea; and 
         R 2  is selected from the group consisting of alkyne, amine, protected amine, azide, hydrazide, aryl, heteroaryl, cycloalkyl, cycloalkenyl, cycloalkylnyl, heterocyclyl, hydroxy, carboxyl, halo, alkoxy, maleimide, —C(O)H, —C(O)OR 8 , —OS(O 2 )R 9 , thiol, biotin, oxyamine, and haloalkyl, wherein R 8  and R 9  are independently selected from the group consisting of alkyl, haloalkyl, aryl, haloaryl, N-succinimidyl, and N-succinimidyl ester; and wherein X −  is a counterion, and n is an integer from 0 to 20. 
       
     
     
         12 . The conjugate of  claim 11 , wherein the transition metal complex and biomolecular binding agent are coupled via click chemistry. 
     
     
         13 . The conjugate of  claim 11 , wherein M is a platinum group metal. 
     
     
         14 . The conjugate of  claim 11 , wherein the transition metal complex has an absorption spectrum in the visible region of the electromagnetic spectrum. 
     
     
         15 . The conjugate of  claim 11 , wherein the conjugate is cell permeable. 
     
     
         16 . The conjugate of  claim 11 , having aqueous solubility of 1 μM to 150 μm at 0.2% DMSO in pure water. 
     
     
         17 . A system for proximity labeling comprising:
 a protein labeling agent; and   a transition metal catalyst, wherein the transition metal catalyst has electronic structure permitting electron transfer to the protein labeling agent to provide a reactive intermediate, and wherein the transition metal catalyst is of Formula I:   
       
         
           
           
               
               
           
         
         wherein M is a transition metal; 
         wherein A, D, E, G, Y and Z are independently selected from C and N; 
         wherein R 3 -R 7  each represent one to four optional ring substituents, each of the one to four optional ring substituents independently selected from the group consisting of alkyl, heteroalkyl, haloalkyl, haloalkenyl, halo, hydroxy, alkoxy, amine, amide, ether, —C(O)O − , —C(O)OR 8 , and —R 9 OH, wherein R 8  is selected from the group consisting of hydrogen and alkyl, and R 9  is alkyl; 
         wherein R 1  is selected from the group consisting of a direct bond, alkylene, alkenylene, cycloaklylene, cycloalkenylene, arylene, heteroalkylene, heteroalkenylene, heterocyclene, and heteroarylene; 
         wherein L is a linking moiety selected from the group consisting of amide, ester, sulfonamide, sulfonate, carbamate, and urea; and 
         R 2  is selected from the group consisting of alkyne, amine, protected amine, azide, hydrazide, aryl, heteroaryl, cycloalkyl, cycloalkenyl, cycloalkylnyl, heterocyclyl, hydroxy, carboxyl, halo, alkoxy, maleimide, —C(O)H, —C(O)OR 8 , —OS(O 2 )R 9 , thiol, biotin, oxyamine, and haloalkyl, wherein R 8  and R 9  are independently selected from the group consisting of alkyl, haloalkyl, aryl, haloaryl, N-succinimidyl, and N-succinimidyl ester; and wherein X −  is a counterion, and n is an integer from 0 to 20. 
       
     
     
         18 . The system of  claim 17 , wherein the electron transfer originates from an excited state of the catalyst electronic structure. 
     
     
         19 . The system of  claim 18 , wherein the electron transfer originates from a triplet state of the catalyst electronic structure. 
     
     
         20 . The system of  claim 17 , wherein the reactive intermediate has a diffusion radius of 1-500 nm. 
     
     
         21 . The system of  claim 20 , wherein the diffusion radius is 1-10 nm. 
     
     
         22 . The system of  claim 17 , wherein the transition metal catalyst is coupled to a biomolecular binding agent. 
     
     
         23 . The system of  claim 22 , wherein the biomolecular binding agent comprises a peptide, protein, sugar, small molecule or nucleic acid. 
     
     
         24 . The system of  claim 22 , wherein the transition metal complex and biomolecular binding agent are coupled via click chemistry. 
     
     
         25 . The system of  claim 17 , wherein the protein labeling agent is a diazirine. 
     
     
         26 . The system of  claim 25 , wherein the diazirine comprises a molecular marker. 
     
     
         27 . The system of  claim 25 , wherein the reactive intermediate is a carbene. 
     
     
         28 . The system of  claim 22 , wherein the transition metal catalyst is cell permeable. 
     
     
         29 . A method of proximity labeling comprising:
 providing a protein labeling agent and conjugate comprising a transition metal catalyst coupled to a biomolecular binding agent;   activating the protein labeling agent to a reactive intermediate with the transition metal catalyst; and   coupling the reactive intermediate to a protein in a cellular environment, wherein the transition metal complex is of Formula I:   
       
         
           
           
               
               
           
         
         wherein M is a transition metal; 
         wherein A, D, E, G, Y and Z are independently selected from C and N; 
         wherein R 3 -R 7  each represent one to four optional ring substituents, each of the one to four optional ring substituents independently selected from the group consisting of alkyl, heteroalkyl, haloalkyl, haloalkenyl, 3halo, hydroxy, alkoxy, amine, amide, ether, —C(O)O − , —C(O)OR 8 , and —R 9 OH, wherein R 8  is selected from the group consisting of hydrogen and alkyl, and R 9  is alkyl; 
         wherein R 1  is selected from the group consisting of a direct bond, alkylene, alkenylene, cycloaklylene, cycloalkenylene, arylene, heteroalkylene, heteroalkenylene, heterocyclene, and heteroarylene; 
         wherein L is a linking moiety selected from the group consisting of amide, ester, sulfonamide, sulfonate, carbamate, and urea; and 
         R 2  is selected from the group consisting of alkyne, amine, protected amine, azide, hydrazide, aryl, heteroaryl, cycloalkyl, cycloalkenyl, cycloalkylnyl, heterocyclyl, hydroxy, carboxyl, halo, alkoxy, maleimide, —C(O)H, —C(O)OR 8 , —OS(O 2 )R 9 , thiol, biotin, oxyamine, and haloalkyl, wherein R 8  and R 9  are independently selected from the group consisting of alkyl, haloalkyl, aryl, haloaryl, N-succinimidyl, and N-succinimidyl ester; and wherein X −  is a counterion, and n is an integer from 0 to 20. 
       
     
     
         30 - 45 . (canceled)